MOLECULAR MAPPING OF MICROBIAL COMMUNITIES AT THE HOST-PATHOGEN INTERFACE BY MULTI-MODAL 3-DIMENSIONAL IMAGING MASS SPECTROMETRY

PROJECT SUMMARY Cellular interactions with the environment form the basis of health and disease for all organisms. Exposureto nutrients, toxins, and neighboring cells trigger coordinated molecular responses that impact cell function andmetabolism in a beneficial, adaptive, or detrimental manner. Although the benefits of multicellularity for theformation of complex tissue structures or the function of entire organ systems has been long appreciated, it hasonly recently been understood that microbial inhabitants of vertebrates also have a tremendous impact on hostcell function and dysfunction. Despite this, an understanding of these interactions has not moved beyond simpleassociations, and there are virtually no molecular technologies available that adequately define how a complexmicrobial ecosystem impacts host cell function, or how the host response to microbial colonization affects thebacterial community. This gap in knowledge is striking when one considers the broad and significant impact thatmicrobes have on human health. In this application, we propose to expressly fill this knowledge gap throughdevelopment of a novel multimodal imaging pipeline that will provide 3-dimensional information on the molecularheterogeneity of microbial communities and the immune response at the host-pathogen interface. This proposal combines our expertise in immunology, infection biology, mass spectrometry, small animalimaging, machine learning, and computer vision to develop an integrated multimodal visualization method forstudying infectious disease. Our unique approach will computationally combine ultra-high speed (~50px/s)MALDI-TOF images, ultra-high mass resolution (>200,000 resolving power) MALDI FTICR IMS, metal imagingby LA-ICP-IMS, high-spatial resolution optical microscopy, and MR imaging using data-driven image fusion. Thisstrategy will enable 3-D molecular images to be generated for thousands of elements, metabolites, lipids, andproteins with an unprecedented combination of chemical specificity and spatial fidelity more than 50x faster thanis currently possible. We will use this next-generation imaging capability to (i) define the heterogeneous microbialsubpopulations throughout the 3-D volume of a S. aureus community, (ii) uncover the host molecules that formthe abscess and accumulate to restrict microbial growth in murine models, and (iii) elucidate molecular markersthat differentiate in vivo biofilms at the host-pathogen interface, between abscesses at various stages ofprogression, and under distinct degrees of nutrient stress. These studies will uncover new targets for therapeuticintervention and the techniques developed as a result of this proposal will be broadly applicable to allphysiologically relevant processes, profoundly impacting biomedical research.